1. Field of the Invention
The invention pertains to the field of cell lines derived from insects. More particularly, the invention pertains to improved cell lines that are susceptible to baculovirus infection and are useful for replicating such viruses, and are useful for gene expression using a baculovirus expression system.
2. Description of Related Art
Insect cell culture has been used broadly in insect virology research since the first insect cell line was established in 1962 (Grace, T. D. C., Establishment of four strains of cells from insect tissue grown in vitro, Nature, 195:788–789). The general use of tissue cell lines for the culture or replication of pathogenic microorganisms is well established, and production of viral insecticides in cell culture has many advantages over their cultivation in vivo ((Weiss, S. A. and J. L. Vaughn, Cell culture methods for large-scale propagation of baculovirus, in “The Biology of Baculoviruses,” vol. II “Practical Application for Insect Control” (Granados and Federici, Eds., CRC Press, Boca Raton, Fla.), pp. 63–87 (1986)) and (Granados et al., Production on viral agents in invertebrate cell cultures, in “Biotechnology in Invertebrate Pathology and Cell Culture” (Maramorosch, K., Ed., Academic Press, San Diego/NY), pp. 167–181, (1987)). However, specific microorganisms cannot be cultured in all cell lines, even in all cell lines from the same order. Vaughn, in Invertebrate Tissue Culture, Research Applications (Academic Press, NY/London, 1976, pp. 295–303) discusses the development of insect cell lines and notes that, for example, cell lines from Heliothis zea are not capable of complete replication of the nuclear polyhedrosis virus obtained from Heliothis zea itself.
In recent years, baculovirus expression vectors have been widely used as vectors for foreign gene expression in insect cells (Luckow, V. A. and M. D. Summers, High level expression of non-fused foreign genes with Autographa californica nuclear polyhedrosis virus expression vectors, Virology, 170:311–39 (1988)). Therefore, insect cell culture has become more important as a potentially attractive system for producing viral insecticides and expressing foreign gene products of interest in the areas of biology, medicine, and agriculture.
Cell lines from Trichoplusia ni eggs have been established and infected ((Rochford et al., Establishment of a cell line from embryos of the Cabbage Looper, Trichoplusia ni (Hubner), In Vitro 20: 823–825 (1984)) and (Granados et al., Replication of the Trichoplusia ni Granulosis and Nuclear Polyhedrosis Viruses in cell cultures, Virology 152: 472–476 (1986))), however, until fairly recently, a Trichoplusia ni embryonic cell line which is highly susceptible to numerous baculoviruses and efficiently supports replication of baculoviruses had not been established. Rochford et al. (1984) developed a Trichoplusia ni egg cell line (IPLB-TN-R) that is susceptible to only one of six baculoviruses tested, the Autographa californica multiply-enveloped nuclear polyhedrosis virus (AcMNPV). In addition, AcMNPV polyhedra production in the IPLB-TN-R cell line occurs later than desirable (beginning at 18 and 39 hours post infection), indicating an inefficient baculovirus replicating cell line. The BTI-TN-5B1-28 embryonic cell line reported by Granados et al. (1986) is moderately susceptible to infection by AcMNPV and Trichoplusia ni singly-enveloped nuclear polyhedrosis virus (TnSNPV).
U.S. Pat. Nos. 5,298,418, 5,300,435, 5,686,305, and 6,403,375 disclose several insect cell lines and the methods used for the establishment and development of novel insect cell lines; the complete disclosures of each of these patents are hereby incorporated herein by reference in their entirety.
BTI-TN-5B1-4 (sold by Invitrogen under the trade name “High 5” cells), as well as IPLB-SF 21 and its clone (Sf9), are the most widely used insect cell lines for the baculovirus expression vector system (Granados et al., J. Invertebr. Pathol., 1994, 64, 260–266; O'Reilly et al., 1992, Baculovirus expression vectors, A laboratory manual, W. H. Freeman and Company, NY). In most instances, High 5 cells provide superior production of recombinant proteins compared to Sf9 cells (Shuler et al., 1995, Baculovirus Expression Systems and Biopesticides, Wiley-Liss Inc., NY.). This high productivity is more evident in low passage culture of High 5 cells in comparison with high passage cells (Donaldson and Shuler, 1998, Biotechnol. Prog., 14, 543–547). This suggests that High 5 cells may be susceptible to detrimental effects associated with long term culturing.